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WO1994026414A1 - Recipient de reaction pour essai par liaison specifique et procede d'utilisation - Google Patents

Recipient de reaction pour essai par liaison specifique et procede d'utilisation Download PDF

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Publication number
WO1994026414A1
WO1994026414A1 PCT/US1994/004849 US9404849W WO9426414A1 WO 1994026414 A1 WO1994026414 A1 WO 1994026414A1 US 9404849 W US9404849 W US 9404849W WO 9426414 A1 WO9426414 A1 WO 9426414A1
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Prior art keywords
chamber
nucleic acid
analyte
channel
sample
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PCT/US1994/004849
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English (en)
Inventor
Edwin F. Ullman
Benjamin R. Irvin
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Syntex (U.S.A.) Inc.
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Publication of WO1994026414A1 publication Critical patent/WO1994026414A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/50273—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the means or forces applied to move the fluids
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502738—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by integrated valves
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/505—Containers for the purpose of retaining a material to be analysed, e.g. test tubes flexible containers not provided for above
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C1/00—Magnetic separation
    • B03C1/005—Pretreatment specially adapted for magnetic separation
    • B03C1/01—Pretreatment specially adapted for magnetic separation by addition of magnetic adjuvants
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0825—Test strips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control
    • B01L2300/1838—Means for temperature control using fluid heat transfer medium
    • B01L2300/185—Means for temperature control using fluid heat transfer medium using a liquid as fluid
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0406—Moving fluids with specific forces or mechanical means specific forces capillary forces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/043—Moving fluids with specific forces or mechanical means specific forces magnetic forces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0481—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure squeezing of channels or chambers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0633—Valves, specific forms thereof with moving parts
    • B01L2400/0655—Valves, specific forms thereof with moving parts pinch valves
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0677—Valves, specific forms thereof phase change valves; Meltable, freezing, dissolvable plugs; Destructible barriers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0677—Valves, specific forms thereof phase change valves; Meltable, freezing, dissolvable plugs; Destructible barriers
    • B01L2400/0683—Valves, specific forms thereof phase change valves; Meltable, freezing, dissolvable plugs; Destructible barriers mechanically breaking a wall or membrane within a channel or chamber
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0688—Valves, specific forms thereof surface tension valves, capillary stop, capillary break
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C2201/00—Details of magnetic or electrostatic separation
    • B03C2201/18—Magnetic separation whereby the particles are suspended in a liquid
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C2201/00—Details of magnetic or electrostatic separation
    • B03C2201/26—Details of magnetic or electrostatic separation for use in medical or biological applications

Definitions

  • This invention relates to reaction containers in which nucleic acids are amplified and detected without exposing the nucleic acid to the environment or the environment to amplified nucleic acid.
  • Nucleic acid hybridization has been employed for investigating the identity and establishing the presence of nucleic acids. Hybridization is based on complementary base pairing. When complementary single stranded nucleic acids are incubated together, the complementary base sequences pair to form double stranded hybrid molecules.
  • ssDNA single stranded deoxyribonucleic acid
  • RNA ribonucleic acid
  • nucleic acid hybridization has great potential in diagnosing disease states associated with unique nucleic acid sequences. These unique nucleic acid sequences may result from genetic or environmental change in DNA by insertions, deletions, point mutations, or by acquiring foreign DNA or RNA by means of infection by bacteria, molds, fungi, and viruses. Nucleic acid hybridization has, until now, been employed primarily in academic and industrial molecular biology laboratories.
  • nucleic acid hybridization as a diagnostic tool in clinical medicine is limited because of the frequently very low concentrations of disease related DNA or RNA present in a patient's body fluid or tissue and the unavailability of a sufficiently sensitive method of nucleic acid hybridization analysis.
  • PCR polymerase chain reaction
  • the two different PCR primers which anneal to opposite strands of the DNA, are positioned so that the polymerase catalyzed extension product of one primer can serve as a template strand for the other, leading to the accumulation of a discrete double stranded fragment whose length is defined by the distance between the 5' ends of the oligonucleotide primers.
  • -Another method that has recently been described is an amplification of a single stranded polynucleotide using a single polynucleotide primer.
  • the single stranded polynucleotide that is to be amplified contains two non-contiguous sequences that are complementary to one another and, thus, are capable of hybridizing together to form a stem-loop structure.
  • This single stranded polynucleotide may be already part of a polynucleotide analyte or may be created as the result of the presence of a polynucleotide.
  • LCR ligase chain reaction
  • NASBA nucleic acid sequence based amplification
  • Another method for amplifying nucleic acids is the Q-beta-replicase method, which relies on the ability of Q-beta-replicase to amplify a specific RNA substrate exponentially and is used as a label to detect binding rather than a method to create more target nucleic acid.
  • nucleic acid probes One method for detecting nucleic acids is to employ nucleic acid probes.
  • One method utilizing such probes is described in U.S. Patent No. 4,868,104, the disclosure of which is incorporated herein by reference.
  • a nucleic acid probe may be, or may be capable of being, labeled with a reporter group or may be, or may be capable of becoming, bound to a support.
  • Detection of signal depends upon the nature of the label or reporter group. If the label or reporter group is an enzyme, additional members of the signal producing system would include enzyme substrates and so forth.
  • the product of the enzyme reaction is preferably a luminescent product, or a fluorescent or non-fluorescent dye, any of which can be detected spectrophotometrically, or a product that can be detected by other spectrometric or electrometric means.
  • the label is a fluorescent molecule
  • the medium can be irradiated and the fluorescence determined. Where the label is a radioactive group, the medium can be counted to determine the radioactive count.
  • the amplification of nucleic acids has been carried out in stoppered containers to obtain the desired number of copies. Then, the container is opened and the amplification medium is withdrawn and transferred to a detection apparatus. Alternatively, the reagents used for detection are added to the container used for the amplification and the detection is carried out in the same container.
  • a technique is unsatisfactory for convenient and widespread use of amplification technology because aerosols are produced in the act of opening the containers and during the transfer of fluids. Such aerosols contain a few molecules of the amplified nucleic acid, which are released into the environment. Normally, such few molecules in the environment are not of great concern.
  • a containment cuvette for amplification of nucleic acids has been disclosed.
  • the cuvette and its method of use are designed to prevent amplified nucleic acid from being released into the atmosphere.
  • the need still exists for devices and methods for carrying out assays that avoid false positives caused by cross-contamination of samples, avoid handling of liquid reagents, are* 5 preferably homogeneous and are automatable with relatively simple instrumentation.
  • a containment cuvette for conducting PCR is disclosed in European Patent Application publication number 0 381 501 (Schnipelsky, et al.) .
  • Detection reagents are either pre-incorporated into compartments in the cuvette or are added after amplification. In the latter situation a check valve prevents amplified nucleic acid from being released. Transfer of liquids between compartments is achieved by the the use of flexible compartment walls and an external pressure source or by pistons that are part of the cuvette and operate on the compartments as a piston within a piston chamber.
  • a device for processing biological specimens for analysis of nucleic acids is described in U.S. Patent No. 5,188,963. The device has a hinged compartment facilitating automation of DNA- and RNA-based diagnostics and genetic surveillance and detection. Specimens are embedded in a matrix in the carrier. The matrix is then treated by one or more of the techniques such as amplification, electrophoresis, and hybridization as selected for the desired analysis and then the sample is treated to detect the
  • PCR A process for amplifying, detecting and/or cloning nucleic acid sequences otherwise referred to as PCR is disclosed in U.S. Patent No ⁇ . 5,008,182, 4,965,188, 4,800,159, 4,683,195 and 4,683,202. Sequence polymerization by PCR is described by Saiki, et al., (1986) Science, 230 : 1350-1354.
  • U.S. Patent Application Serial No. 07/555,323 filed July 19, 1990 discloses methods for producing a polynucleotide for use in single primer amplification.
  • U.S. Patent Application Serial No. 07/555,968 filed July 19, 1990 describes a method for producing a molecule containing an intramolecular base-pair structure.
  • U.S. Patent Application Serial No. 07/776,538 filed October 11, 1991 discloses methods for producing a polynucleotide for use in single primer amplification.
  • U.S. Patent Application Serial No. 07/923,079 filed July 31, 1992 describes a method for introducing sequences at the 3' end of polynucleotides. The disclosures of these six applications are incorporated herein by reference in their entirety.
  • LCR ligase chain reaction
  • NASBA nucleic acid sequence based amplification
  • Q-beta-replicase amplification of RNA is also discussed in European Patent Applications Nos. 439,182 (Backman I) and 473,155 (Backman II) .
  • the method comprises treating the reaction mixture suspected of containing the amplified nucleic acid analyte under conditions such that the analyte, if present, causes a photosensitizer and a chemiluminescent compound to come into " close proximity.
  • the photosensitizer generates singlet oxygen and activates the chemiluminescent compound when it is in close proximity.
  • the activated chemiluminescent compound subsequently produces light.
  • the amount of light produced is related to the amount of analyte in the medium.
  • the present invention includes devices and methods for using the devices for conducting amplifications of nucleic acids.
  • One device in accordance with the present invention comprises (a) a sample receiving chamber, (b) a plurality of additional chambers, at least one of the additional chambers containing reagents for carrying out an assay for the determination of a nucleic acid, (c) means for detecting a signal generated by the reagents, (d) means for permitting fluid communication between the additional chambers, (e) means for introducing a sample into the device, and (f) means for separating the nucleic acid analyte from the remainder of the sample and introducing the nucleic acid analyte into one of the additional chambers, which means comprises suspendible particles.
  • Another embodiment in accordance with the present invention is a device for amplifying and detecting a nucleic acid analyte comprising: (a) a port for introducing a sample suspected of containing a nucleic acid analyte into the device, (b) a first chamber, for receiving the sample, in fluid communication with the port, wherein fluid communication between the port and the first chamber is capable of being sealed off, (c) a channel, containing a liquefiable gel, in fluid communication with the first chamber, (d) a second chamber containing reagents for amplifying the nucleic acid analyte, wherein fluid communication between the second chamber and the channel is prevented by a temporary seal, and (e) a third chamber, wherein fluid communication between the second chamber and the third chamber is prevented by a temporary seal.
  • Another embodiment in accordance with the present invention is a method for amplifying and detecting a nucleic acid analyte using a sealable device.
  • the method comprises the steps of: (a) introducing a sample suspected of containing the nucleic acid analyte into a first chamber of the device preceding or following combining the sample with particles capable of binding the nucleic acid analyte, (b) transporting the particles from the first chamber through a channel containing air or a liquefiable gel into a second chamber, wherein the channel provides fluid communication between the chambers, (c) sealing off the channel, (d) establishing fluid communication between the second chamber and a third chamber of the device, (e) introducing into the third chamber a liquid medium for reagents for amplifying the nucleic acid analyte, when such liquid medium is not already present, (f) transporting the analyte into the third chamber, (g) subjecting the medium to conditions for amplifying nucleic acids, (h)
  • kits for conducting an assay for a nucleic acid analyte comprise in packaged combination a device as described above.
  • the kits can further comprise in packaged combination particles capable of binding the nucleic acid analyte or reagents for amplification or detection of nucleic acids if not present in the device.
  • FIG. 1 is a plan view of a device in accordance with the present invention.
  • Fig. 2 is a plan view of the device of Fig. 1 with its top portion folded on itself.
  • Fig. 3 is a plan view of another device in accordance with the present invention.
  • Fig. 4 is a cross-sectional view of the device of Fig. 3.
  • Fig. 5 is a plan view of another device in accordance with the present invention.
  • Fig. 6 is a plan view of another device in accordance with the present invention.
  • the present invention provides devices and methods for conducting nucleic acid amplification reactions.
  • the present invention is particularly useful for the homogeneous assay of nucleic acids used in conjunction with an amplification procedure.
  • the present devices differ from known devices by, inter alia, the presence of a liquefiable gel or an air gap for transporting the sample bound to particles within a portion of the device.
  • the present device can be utilized to conduct homogeneous nucleic acid assays, that is, assays not requiring a separation step.
  • a further distinction is that some of the present devices have a tandem arrangement of channels and chambers.
  • the present devices do not include means for separating assay components in a detection chamber as required in some of the prior art devices. .• ⁇
  • Nucleic acid or polynucleotide analyte a compound or composition to be measured that is a polymeric nucleotide or a portion of a polymeric nucleotide, which in the intact natural state can have about 200 to 500,000 or more nucleotides and in an isolated state can have about 30 to 50,000 or more nucleotides, usually about 100 to 20,000 nucleotides, more frequently 500 to 10,000 nucleotides. It is thus obvious that isolation of the analyte from the natural state often results in fragmentation of the polymeric nucleotide.
  • the polynucleotide analytes include nucleic acids from any source in purified or unpurified form including DNA (dsDNA and ssDNA) and RNA, including t-RNA, m-RNA, r-RNA, mitochondrial DNA and RNA, chloroplast DNA and RNA, DNA-RNA hybrids, or mixtures thereof, genes, chromosomes, plasmids, the genomes of biological material such as microorganisms, e.g., bacteria, yeasts, viruses, viroids, molds, fungi, plants, animals, humans, and fragments thereof, and the like.
  • the polynucleotide analyte can be only a minor fraction of a complex mixture such as a biological sample.
  • the analyte can be obtained from various biological materials by procedures well-known in the art. Some examples of such biological material by way of illustration and not limitation are disclosed in Table I of U.S. Patent Application Ser. No. 07/923,079 filed July 31, 1992, which Table I is incorporated herein by reference.
  • the polynucleotide analyte may be treated to cleave the analyte to obtain a polynucleotide fragment that contains a target polynucleotide sequence. Such cleaving treatments may be accomplished, for example, by shearing or by treatment with a restriction endonuclea ⁇ e or other site specific chemical cleavage method.
  • polynucleotide analyte can be used in its isolated state without further cleavage.
  • the cleaved and uncleaved polynucleotide fragments may each be referred to herein as a v polynucleotide analyte.
  • the polynucleotide analyte, or a cleaved fragment obtained from the polynucleotide analyte may be at least partially denatured or single stranded or treated to render it denatured or single stranded.
  • treatments are well-known in the art and include, for instance, heat or alkali treatment.
  • double stranded DNA when heated at 90-100° C. for a period of 10-20 seconds or more, produces denatured material.
  • Sample the material suspected of containing the nucleic acid analyte.
  • samples include biological fluids such as blood, serum, plasma, sputum, lymphatic fluid, semen, vaginal mucus, fece ⁇ , urine, spinal fluid, and the like; biological tissue such as hair and skin; and so forth.
  • Other samples include cell cultures and the like, plants, food, forensic samples such as paper, fabrics and scrapings, water, sewage, medicinals, etc..
  • the sample may be pretreated with reagents to liquefy the sample and release the nucleic acids from binding substances. Such pretreatments are well-known in the art.
  • Polynucleotide primer a polynucleotide, usually a synthetic polynucleotide, usually single stranded and selected in view of the known sequence of the polynucleotide analyte depending on the type of amplification to be conducted.
  • the polynucleotide primer(s) are usually comprised of a sequence of at least 10 nucleotides, preferably, 20 to 90 nucleotides, more preferably, 24 to 64 nucleotides.
  • polynucleotide primers can be obtained by biological synthesis or by chemical synthesis. For short sequences (up to about 100 nucleotides) chemical synthesis is frequently more economical as compared to biological synthesis. For longer sequences standard replication methods employed in molecular biology can be used such as the use of M13 for single stranded DNA as described by J. Messing, Methods Enzymol(1983) 101 : 20-78.
  • in vitro enzymatic methods may be used such as polymerase catalyzed reactions.
  • T7 RNA polymerase and a suitable DNA template can be used.
  • DNA polymerase chain reaction (PCR) and single primer amplification are convenient.
  • Deoxynucleoside triphosphates deoxynucleosides having a 5' -triphosphate substituent.
  • the deoxynucleosides are pentose sugar derivatives of nitrogenous bases of either purine or pyrimidine derivation, covalently bonded to the 1' -carbon of the pentose sugar.
  • the purine bases include adenine(A) , guanine(G) , inosine, and derivatives and analogs thereof.
  • the pyrimidine bases include cytosine (C) , thymine (T) , uracil (U) , and derivatives and analogs thereof.
  • the derivatives and analogs include any substrate of a polydeoxynucleotide polymerase that can be incorporated into a polynucleotide through catalysiB by ⁇ uch enzyme.
  • the derivates and analogs are exemplified by those that are recognized and polymerized by the enzyme in a similar manner to the underivitized nucleoside triphosphates. Examples of such derivatives or analogs by way of illustration and not limitation are those that are modified with a reporter group, biotinylated, a ine modified, radiolabeled, alkylated, and the like and also include phosphorothioate, phosphite, ring atom modified derivatives, unnatural bases, and the like.
  • the reporter group can be a fluorescent group such as fluorescein, a chemiluminescent group such a ⁇ luminol, a terbium chelator such as N- (hydroxyethyl) ethylenediaminetriacetic acid that is capable of detection by delayed fluorescence, and the like.
  • Amplification of nucleic acids or polynucleotides any method that results in the formation of one or more copies of a nucleic acid or polynucleotide molecule, usually a nucleic acid or polynucleotide analyte, present in a medium.
  • One such method for the enzymatic amplification of specific double stranded sequences of DNA is known a ⁇ the polymerase chain reaction (PCR), as described above.
  • This in vitro amplification procedure is ba ⁇ ed on repeated cycles of denaturation, oligonucleotide primer annealing, and primer extension by thermophilic template dependent polynucleotide polymerase, resulting in the exponential increase in copies of the desired sequence of the polynucleotide analyte flanked by the primers.
  • the two different PCR primers which anneal to opposite strands of the DNA, are positioned ⁇ o that the polymerase catalyzed extension product of one primer can serve a ⁇ a template strand for the other, leading to the accumulation of a discrete double stranded fragment whose length is defined by the distance between the 5' end ⁇ of the oligonucleotide primer ⁇ .
  • the single ⁇ tranded polynucleotide that is to be amplified contains two non-contiguous sequence ⁇ that are complementary to one another and, thu ⁇ , are capable of hybridizing together to form a stem- loop structure.
  • This single ⁇ tranded polynucleotide may be already part of a polynucleotide analyte or may be created a ⁇ the re ⁇ ult of the presence of a polynucleotide.
  • LCR ligase chain reaction
  • NASBA nucleic acid sequence ba ⁇ ed amplification
  • Another method for amplifying a specific group of nucleic acids is the Q-beta-replicase method, which relie ⁇ on the ability of Q-beta- replica ⁇ e to amplify it ⁇ RNA ⁇ ub ⁇ trate exponentially.
  • the polydeoxynucleotide polymerase is a template dependent polydeoxynucleotide polymerase and utilizes the deoxynucleoside triphosphates as building blocks for extending the 3' end of the polynucleotide primer to provide a sequence complementary with a single stranded polynucleotide sequence.
  • the catalysts are enzymes, ⁇ uch as DNA polymerases, for example, prokaryotic DNA polymerase (I, II, or III) , T4 DNA polymerase, T7 DNA polymerase, Klenow fragment, reverse transcriptase.
  • Vent DNA polymerase (Vent is a trademark of New England BioLabs, Beverly, MA) , Pfu DNA polymerase, Ta ⁇ DNA polymerase, and the like, derived from any source such as cells, bacteria, for example, E. coli. plants, animals, virus, thermophilic bacteria, and so forth.
  • reverse transcriptase is used as at least one of the polynucleotide polymerases to facilitate extension of the primer along the complementary strand ⁇ of the polynucleotide analyte.
  • Hybridization and binding -- in the context of nucleotide ⁇ equences these terms are used interchangeably herein.
  • the ability of two polynucleotide sequence ⁇ to hybridize with each other is based in a large part on the degree of complementarity of the two polynucleotide sequences, which in turn is based on the fraction of matched complementary nucleotide pairs.
  • the more nucleotide ⁇ in a given sequence that are complementary to another sequence the more stringent the conditions can be for hybridization and the more specific will be the binding of the two sequence ⁇ .
  • Another factor to be con ⁇ idered i ⁇ the nature of the nucleotide pair ⁇ that are oppo ⁇ ite in the two strand ⁇ .
  • Some nucleotide pair ⁇ , such a ⁇ G and C have greater binding affinities for one another than do other pairs.
  • Increased stringency is achieved by elevating the temperature, increasing the ratio of cosolvents, lowering the salt concentration, and the like.
  • Homologous or substantially identical In general, two polynucleotide sequence ⁇ that are identical, or at least can each hybridize to the same polynucleotide ⁇ equence, are homologous.
  • the two sequence ⁇ are h omologous or substantially identical where the sequences each have at least 90%, preferably 100%, of the same or analogous base sequence where thymine (T) and uracil (U) are con ⁇ idered the same.
  • T thymine
  • U uracil
  • the ribonucleotides A, U, C and G are taken a ⁇ analogous to the deoxynucleotides dA, dT, dC, and dG, re ⁇ pectively.
  • Homologous ⁇ equences can both be DNA or one can be DNA and the other RNA.
  • Complementary--two ⁇ equences are complementary when the sequence of one can bind to the sequence of the other in an anti-parallel sense wherein the 3' end of each sequence bind ⁇ to the 5' end of the other ⁇ equence and, for example, among the natural ba ⁇ es each A, T(U), G, and C of one sequence i ⁇ then aligned with a T(U) , A, C, and G, respectively, of the other ⁇ equence.
  • sbp member one of two different molecule ⁇ , having an area on the surface or in a cavity that specifically binds to and is thereby defined as complementary with a particular spatial and polar organization of the other molecule.
  • the members of the ⁇ pecific binding pair are referred to a ⁇ ligand and receptor (antiligand) .
  • the ⁇ e may be member ⁇ of an immunological pair such as antigen-antibody, or may be operator-repressor, nuclease-nucleotide, biotin-avidin, hormone ⁇ -hormone receptor ⁇ , nucleic acid duplexes, IgG-protein A, DNA-DNA, DNA-RNA, and the like.
  • Ligand any compound for which a receptor naturally exi ⁇ ts or can be prepared.
  • Receptor any compound or composition capable of recognizing a particular ⁇ patial and polar organization of a molecule, e.g., epitopic or determinant ⁇ ite.
  • Illu ⁇ trative receptors include naturally occurring receptors, e.g., thyroxine binding globulin, antibodies, enzyme ⁇ , Fab fragments, lectins, nucleic acid ⁇ , repressors, protection enzyme ⁇ , protein A, complement component Clq, DNA binding protein ⁇ or ligand ⁇ and the like.
  • Small organic molecule a compound of molecular weight le ⁇ than
  • the small organic molecule can provide a means for attachment of a nucleotide sequence to a label or to a particle.
  • the non-magnetic particle ⁇ are u ⁇ ually diamagnetic or paramagnetic with a magnetic susceptibility (x) of less than 1X10' 5 e uj/Oecm 3 .
  • the non-magnetic particle ⁇ may be organic or inorganic, swellable or non-swellable, porous or non-porous, usually of a density heavier than water, generally about 1.1 to 13, preferably 2 to 10 g/ml.
  • the particles can be, for example, organic an inorganic polymers, latex particles, inorganic powder ⁇ ⁇ uch as silica, magnesium ⁇ ulfate, and alumina; natural polymeric materials, ⁇ ynthetic or modified naturally occurring polymers, such a ⁇ nitrocellulose, cellulose acetate, poly (vinyl chloride) , polyacrylamide, cross linked dextran, agarose, polyacrylate, polyethylene, polypropylene, poly(4-methylbutene) , poly ⁇ tyrene, polymethacrylate, poly(ethylene terephthalate) , nylon, poly(vinyl butyrate) , etc.; either u ⁇ ed by them ⁇ elve ⁇ or in conjunction with other materials; glass available as Bioglass, ceramics, metals, and the like.
  • the magnetic particles are intrinsically magnetically responsive or have been rendered magnetic by, for example, attachment to a magnetically responsive substance or by incorporation of such substance into the particles.
  • the magnetic particles can be paramagnetic, ferromagnetic, or superparamagnetic, usually paramagnetic or superparamagnetic and have magnetic susceptibilities (x) of at least 5X10' 5 , usually 4X10 e uj/Oecm 3 .
  • Exemplary of the magnetic component of particles that are intrinsically magnetic or magnetically responsive are complex salts and oxides, borides and sulfides of iron, cobalt, nickel and rare earth elements having high magnetic susceptibility, e.g., hematite, ferrite and so forth.
  • the magnetic component of other ⁇ uch particles includes pure metals or alloys comprising one or more of these elements.
  • Binding of sbp member ⁇ to particles may be accomplished by well-known techniques, commonly available in the literature. See, for example,
  • Label or reporter group or reporter molecule a member of the signal producing system.
  • the label or reporter group or molecule is conjugated to or becomes bound to a polynucleotide probe or a polynucleotide primer and is capable of being detected directly, or indirecting by being bound through a specific binding reaction, to a detectable sub ⁇ tance.
  • Label ⁇ able to be detected indirectly include polynucleotide ⁇ such a ⁇ a polynucleotide primer or a specific polynucleotide sequence that can act as a ligand for a complementary polynucleotide or provide a template for amplification or ligation or act a ⁇ a ligand such a ⁇ for a repressor protein; hapten ⁇ ; antibodie ⁇ ; receptors such as avidin; ligands such as biotin and the like.
  • Labels able to be detected directly may be isotopic or nonisotopic, u ⁇ ually non-i ⁇ otopic, and can be a catalyst, ⁇ uch as an enzyme, ribozyme, a substrate for a replicase such as QB replicase, promoter, dye, fluore ⁇ cent molecule, chemiluminescer, coenzyme, enzyme sub ⁇ trate, radioactive group, a particle such as latex or carbon particle, metal sol, crystallite, liposome, cell, etc., which may or may not be further labeled with a dye, catalyst or other detectible group, and the like.
  • a catalyst ⁇ uch as an enzyme, ribozyme, a substrate for a replicase such as QB replicase, promoter, dye, fluore ⁇ cent molecule, chemiluminescer, coenzyme, enzyme sub ⁇ trate, radioactive group, a particle such as latex or carbon particle, metal sol, crystallite, liposome, cell, etc., which may or
  • the label is a member of a ⁇ ignal producing ⁇ y ⁇ tem and can generate a detectable signal either alone or together with other member ⁇ of the signal producing system.
  • the label can be bound directly to a nucleotide ⁇ equence or can become bound thereto by being bound to an sbp member complementary to an sbp member that is bound to a nucleotide sequence.
  • Method ⁇ for binding of labels to nucleotide ⁇ are well-known and described, for example, in U.S Patent No. 4,948,882 (Ruth), U.S. Patent No. 5,082,830 (Brakel, et al.), U.S. Patent No. 4,894,325 (Koser, et al.) and U.S. Patent No. 4,987,065 (Stavrianopolis, et al.) .
  • the signal producing system may have one or more components, at least one component being the label or reporter group.
  • the signal producing ⁇ ystem generates a signal that relate ⁇ to the presence or amount of nucleic acid analyte in a sample.
  • the signal producing system includes all of the reagents required to produce a measurable signal.
  • the label is normally bound to an sbp member complementary to an sbp member that is bound to or part of a nucleotide sequence.
  • components of the signal producing system may be included in a developer solution and can include substrates, enhancers, activators, chemilumine ⁇ cent compound ⁇ , cofactors, inhibitors, scavengers, metal ions, specific binding sub ⁇ tances required for binding of signal generating substances, and the like.
  • Other components of the signal producing system may be coenzyme ⁇ , subBtance ⁇ that react with enzymic product ⁇ , other enzymes and catalyst ⁇ , and the like.
  • the ⁇ ignal producing system provides a signal detectable by external means, such as detection of electromagnetic radiation, de ⁇ irably by visual examination.
  • the signal-producing system is de ⁇ cribed more fully in U.S. Patent Application Serial No. 07/555,323, filed July 19, 1990, the relevant disclosure of which is incorporated herein by reference.
  • buffers will normally be present in the medium, a ⁇ well as stabilizers for the medium and the reaction components.
  • proteins may be included, such as albumins, organic ⁇ olve ⁇ t ⁇ ⁇ uch a ⁇ formamide, quaternary ammonium salts, polyanions ⁇ uch as dextran ⁇ ulfate, ⁇ urfactant ⁇ , particularly non-ionic surfactants, binding enhancers, e.g., polyalkylene glycol ⁇ , or the like.
  • Device 10 has a sample receiving channel 12 with port 14 for introduction of sample into device 10.
  • Channel 12 is in fluid communication with first chamber 16 and with second channel 18, which is in potential fluid communication with second chamber 20.
  • the fluid communication between channel 18 and chamber 20 i ⁇ temporarily prevented by temporary seal 22, thus rendering the fluid communication between channel 18 and chamber 20 potential.
  • Both channel 18 and chamber 20 are filled with a heat meltable or liquifiable gel 24.
  • Chamber 20 is in potential fluid communication with a third channel 26, having a temporary seal 27.
  • Channel 26 is in fluid communication with third chamber 28.
  • Device 10 is constructed such that top portion 30 i ⁇ foldable on itself along line 32, thus permitting channel 12 to be sealed off from the environment as seen in Fig. 2.
  • This i ⁇ by way of example and not limitation. Sealing of the device after sample has been introduced through port 14 may be accompli ⁇ hed by other mean ⁇ ⁇ uch as heat sealing of the port or of channel 12 at a point adjacent to port 14.
  • port 14 may be closed by pinching, stoppering, capping and the like.
  • Port 14 may also comprise a ⁇ elf- ⁇ ealing ela ⁇ tomer.
  • the liquefiable or heat meltable gel i ⁇ a thermally liquefiable gel that may be a poly ⁇ accharide, polyacrylate, p ⁇ lypeptide, polyvinylalcohol, polyether, poly ⁇ iloxane or the like.
  • the thermally liquefiable gel i ⁇ liquefiable at a temperature of about 35 to 105° C, preferably, 45 to 95° C, more preferably, 60 to 70° C. It is important to note that with some gels it is not necessary that the entire gel be liquified to achieve the neces ⁇ ary movement of particle ⁇ with the nucleic acid analyte bound thereto through the gel.
  • the thermally liquefiable gel when liquefied, ha ⁇ a vi ⁇ co ⁇ ity that permit ⁇ movement of the nucleic acid bound to particle ⁇ through the gel; usually a viscosity of about 0.005 to 0.5 poise, preferably 0.01 to 0.05 poise.
  • the mas ⁇ density of the gel may be adjusted by adding to the gel a heavy metal salt, such as cesium chloride, or polyiodinated aromatic compounds such as those sold under the trade name NYCODENZ, in an amount sufficient to achieve the desired density, preferably 1.05 to 1.2 g/cm 3 .
  • the mass density of the gel is greater than that of the sample to ensure that; the sample and the liquified gel are stably stratified, thereby avoiding mixing and sub ⁇ equent contamination of the remainder of the device with the sample.
  • the density or vi ⁇ co ⁇ ity of the gel is greater than that of the sample to prevent the liquified gel and the sample from mixing, thereby avoiding contamination of the remainder of the device with the sample.
  • thermally liquefiable gel by way of example and not limitation, are the following: polysaccharide ⁇ such as agarose; polyacrylates such as polyacrylamide; polypeptides such as gelatin; polyvinylalcohols; polyether ⁇ ; and polysiloxanes.
  • a preferred gel to adjust the density is agarose (0.5% w/v) (e.g., SeaPlaque ® , FMC, Bioproducts, Rockland, ME with a polyiodinated aromatic compound ⁇ uch as Nycodenz ® which is a non-ionic tri-iodinated derivative of benzoic acid with three aliphatic hydrophilic side chains.
  • Nycodenz ® The sy ⁇ tematic name of Nycodenz ® is 5- (n-2,3-dihydroxypropylacetamid ⁇ ) -2,4,6-tri-iodo-n, n'- bi ⁇ (2,3 dihydroxypropyl) i ⁇ ophthalamide. (Nycodenz ® , Nycomed AS, O ⁇ lo, Norway) .
  • the liquefiable gel ⁇ erve ⁇ as the only solvent for the sample.
  • the appropriate channel and chamber of the device can be filled with gel during manufacture of the device by injecting molten gel into the channel or chamber or both. Air pocket formation is preferably avoided.
  • the device may be supported vertically on a rigid backing to prevent the gel from being disrupted as the device is handled during the procedure. The gel i ⁇ then cooled to facilitate ⁇ olidification.
  • the breaking of a frangible ⁇ eal is one means for permitting the establishment of fluid communication between a channel and a chamber or between chambers.
  • Other means for permitting the establishment of fluid communication includes relieving the pressure on a seal produced by pressure on the flexible wall of a channel, applying hydrostatic pressure to a channel sealed by surface tension, charge, and/or Van der Waals forces, unbinding a channel that is sealed as a result of a bend, melting a wax, crystalline, or gel plug in a channel, photodepolymerization of a polymer comprising a gel, etc.
  • Fluid communication between a channel and a chamber or between chambers can be terminated by means for sealing off the fluid communication.
  • Such means can be, for example, a ⁇ eal produced by application of external pres ⁇ ure, a portion of the device foldable on itself as described above for the port, heatsealing, freezing, photopolymerization of a monomer, and so forth.
  • device ⁇ in accordance with the pre ⁇ ent invention are preferably compo ⁇ ed of at least one flexible layer and a second layer that may be flexible or rigid.
  • the device can be manufactured from two sheets of flexible plastic sealed together and having interior blisters or bubbles forming the chambers and channels.
  • the first chamber i ⁇ larger than the other chambers, but need not be.
  • the volume of the sample receiving or first chamber is usually about 0.02 to 2 ml, preferably, 0.1 to 1.0 ml.
  • the volume of each of the remaining chambers is usually independently about 0.02 to 1 ml, preferably, 0.1 to .5 ml.
  • the volume of the liquefiable gel is usually about 0.1 to 3 ml, preferably, 0.2 to 2 ml. Generally, this volume of gel i ⁇ di ⁇ tributed between the first channel and the second chamber, both of which contain the gel.
  • the material for use in manufacturing a device in accordance with the present invention should be thermally stable, provide a moisture barrier, be chemically compatible with the reagents u ⁇ ed for the handling and tran ⁇ fer of ⁇ ample and amplification and detection of amplified nucleic acid and compatible with the variou ⁇ procedure ⁇ such as heatsealing employed in using the device, and be sealable and formable.
  • the material for fabrication of the pre ⁇ ent device should be flexible enough to permit the device to be manipulated during liquid transfer steps involving squeezing of parts of the device to force contents from one area of the device to another.
  • the material should not soften appreciably at temperature ⁇ up to about 100° C.
  • Moi ⁇ ture barrier propertie ⁇ of the material can be quantified by the moisture vapor tran ⁇ mi ⁇ ion rate (MVTR) measured in g-mil/100 sq. in. /24 hour.
  • MVTR at room temperature for the material is le ⁇ than about 0.2, preferably le ⁇ than, 0.05 g- mil/100 ⁇ q in /24 hour.
  • U ⁇ e of lamination of two or more different sheets of plastic film helps to reduce the MVTR values.
  • the thickne ⁇ of the pla ⁇ tic film or ⁇ heet ⁇ used in the manufacture of a device in accordance with the present invention i ⁇ about 1 to 6 mil, preferably, 2 to 4 mil.
  • the ⁇ e values apply to the plastic film as a single material or as a laminate.
  • Suitable pla ⁇ tic ⁇ that may be u ⁇ ed in the present device are, by way of example and not limitation, polyolefins ⁇ uch as polypropylene and low or medium density polyethylene; ethylene-vinyl acetate; polyvinylidene chloride; chlorotrifluoroethane; and the like and laminations of two or more of the above. It is also within the scope of the present invention to use laminates of one or more of the above plastics with a pla ⁇ tic other than that recited specifically above a ⁇ long as the requisite characteristics of the device are obtained. Frequently, the device is comprised of two plastic films, u ⁇ ually heat sealed together.
  • the device may further be mounted on a rigid backing, usually plastic such as polyvinylchloride (PVC) , polycarbonate, nylon, polyethylene, etc., or glas ⁇ or metal.
  • a rigid backing usually plastic such as polyvinylchloride (PVC) , polycarbonate, nylon, polyethylene, etc., or glas ⁇ or metal.
  • PVC polyvinylchloride
  • one plastic film can be sealed directly to a rigid backing, which then usually is. a plastic material.
  • one sheet of plastic used to form the device is formed into bubbles or blisters to make the various channels and chambers of the device. This is usually accomplished by stretching the sheet or film of pla ⁇ tic over or into a mold. Thermoforming is the most common method of accomplishing this molding. The film i ⁇ placed on a forming die that ha ⁇ been cut to create the ⁇ hape ⁇ of the blisters. Vacuum and heat are applied during the thermoforming process. The plastic used in this sheet must flow and thin so that it can be formed into the appropriate shape ⁇ . Laminate ⁇ are particularly suitable for this purpose.
  • the film is formed into the desired shape, it is joined with another film or sheet of plastic or a backing, which may or may not be of the same composition as the first film.
  • Numerous methods are available to join the plastic films together.
  • One such method i ⁇ heatsealing using a hot die.
  • Many apparatus for heatsealing are commercially available.
  • Other methods that are variations of heatsealing include ultrasonic welding, RF sealing and vibration sealing.
  • Heatsealing i ⁇ often carried out u ⁇ ing a laminate with a ⁇ pecific heat ⁇ eal layer on the side to be ⁇ ealed, typically polyethylene or ethylene vinyl acetate.
  • Other method ⁇ for joining the pla ⁇ tic film ⁇ are ⁇ olvent bonding and adhesive bonding.
  • the device is about 2 to 25 cm, preferably, 4 to 15 cm, in length, about 8 to 30 cm, preferably, 10 to 20 mm, in width, and about 0.1 to 4 mm, preferably, 2 to 2 mm, in depth.
  • the material used to make the dies for the forming and heat ⁇ ealing step ⁇ should preferably allow low heat flux into the die, thereby decreasing the gradient across the plastic films. Heat seal temperature can then be reached at lower platen temperatures.
  • the material preferably should be easy to machine and able to hold its shape after many applications of heat and pre ⁇ ure.
  • the material should be slightly compressible under the platen pres ⁇ ure ⁇ employed in the forming and heatsealing step ⁇ .
  • Suitable material ⁇ are printmaking linoleum, e.g., ⁇ uch as manufactured by Speedball Printma ⁇ ter ® (Hunt Mfg. Co., Philadelphia, PA) metal ⁇ uch a ⁇ aluminum; ⁇ ilicone rubber; ga ⁇ ket paper; phenolic compo ⁇ ite; laboratory labeling tape; and the like.
  • the heat ⁇ ealing step i ⁇ conducted at a platen temperature of about 260 to 320, preferably, 280 to 300°F. with a contact time of about 0.2 to 5, preferably, 0.5 to 2 seconds and at a platen pres ⁇ ure of about 200 to 1200, preferably, 600 to 900 pounds per ⁇ quare inch (p ⁇ i) .
  • the heat sealing conditions should be balanced to achieve both a weak frangible seal and a strong seal over the perimeter of the device.
  • the frangible seal is formed in a groove that i ⁇ cut ⁇ hallow in the die.
  • the interface in this groove heats up more ⁇ lowly than the perimeter ⁇ eal.
  • the platen temperature is usually about 290 to 300°C. with a platen pressure of about 600 to 800 p ⁇ i and a contact time of about 0.7 to 0.9 seconds.
  • the platen is lifted before the groove seal reaches melt strength.
  • the frangible seal in the present device should break with an applied force of about 5 to 15 pounds. A balance must be achieved between breaking the frangible seal and breaking the perimeter seal of the device.
  • frangible and perimeter seal ⁇ Another method to achieve the appropriate frangible and perimeter seal ⁇ involve ⁇ interpo ⁇ ing at the point of the frangible seal a substance that interferes with the heatseal. This result ⁇ in a seal at that point that is weaker than the perimeter seal. Upon application of pressure to the channel or chamber containing the frangible seal, the seal breaks more readily than the perimeter seal.
  • Such sub ⁇ tance ⁇ are generally oily or tacky and may be, by way of example and not limitation, a ⁇ olvent-ba ⁇ ed adhe ⁇ ive that retains some elasticity upon evaporation of the solvent such as a mixture of transfer tape adhesive (e.g., 3M Company) and silicone fluid di ⁇ olved in xylene and thinned with methylene chloride (1 part adhe ⁇ ive, 1 part ⁇ ilicone fluid, 2 parts xylene and 2 parts methylene chloride) , rubber cement thinned in methylene chloride (1 part rubber cement and 1 part methylene chloride) , a ⁇ ub ⁇ tance contained in the PAP Pen (Daido Sangyo, Japan), and the like.
  • a ⁇ olvent-ba ⁇ ed adhe ⁇ ive that retains some elasticity upon evaporation of the solvent
  • silicone fluid di ⁇ olved in xylene and thinned with methylene chloride (1 part adhe ⁇
  • FIG. 3 and 4 A bli ⁇ ter pack device in accordance with the pre ⁇ ent invention is depicted in Fig ⁇ . 3 and 4.
  • Device 40 ha ⁇ entry port 42 for introduction of ⁇ ample into device 40.
  • Port 42 is in fluid communication with ⁇ a ple receiving chamber 44.
  • the bottom portion 46 of chamber 44, channel 48 and chamber 50 are in fluid communication and are filled with a heat meltable or liquifiable gel 52.
  • Neck 51 i ⁇ capable of being sealed off by application of pre ⁇ ure.
  • Frangible ⁇ eal 54 in channel 56 prevent ⁇ fluid communication between chamber 50 and chamber 58.
  • Chamber 50 may contain one or more reagent ⁇ for conducting an amplification of nucleic acid ⁇ and chamber 58 may contain one or more reagent ⁇ for the detection of amplified nucleic acid ⁇ .
  • Another embodiment of the pre ⁇ ent invention i ⁇ a method for amplifying and detecting a nucleic acid analyte u ⁇ ing a ⁇ ealable device.
  • the method compri ⁇ e ⁇ the steps of: (a) introducing a sample suspected of containing the nucleic acid analyte into a first chamber of the device preceding or following combining the sample with particles capable of binding the nucleic acid analyte, (b) transporting the particles from the first chamber through a channel containing air or a liquefiable gel into a ⁇ econd chamber, wherein the channel provides fluid communication between the chambers, (c) sealing off the channel, (d) establishing fluid communication between the second chamber and a third chamber of the device, (e) introducing into the third chamber a liquid medium for reagents for amplifying the nucleic acid analyte, when such liquid medium is not already present, (f) transporting the analyte into the third chamber, (g) subjecting the
  • a first step the sample is combined with particles capable of binding the nucleic acid analyte.
  • particle ⁇ capable of binding the nucleic acid analyte can be present in the first chamber 72 of device 70.
  • the sample is introduced into the device through port 74 and top portion 94 is folded along line 92 to seal the port.
  • the particles are capable of binding the nucleic acid analyte by virtue of having bound thereto a polynucleotide sequence capable of hybridizing to, and preferably complementary to, the nucleic acid analyte.
  • the particles can be non-magnetic or magnetic, preferably magnetic.
  • the particles are non-magnetic with a density heavier than water to permit separation from the medium containing the sample through settling and movement of the particles from the fir ⁇ t chamber through channel 76 to the second chamber 78 after breaking of frangible seal 79.
  • the particles should have a density greater than that of the gel. The gel is in lower portion 80 of chamber 72 and in channel 76 of device 70.
  • the ⁇ ample can be combined with magnetic particle ⁇ capable of binding the nucleic acid analyte and moved through the ⁇ ample medium or air, or through liquefied gel if a gel is employed, by application of a magnetic field gradient such a ⁇ by magnet 82 in Fig. 5.
  • a magnetic field gradient such as a ⁇ by magnet 82 in Fig. 5.
  • For magnetic transfer through a gel it is fir ⁇ t nece ⁇ ary to partially or fully liquefy the gel. Usually, this is accomplished by heating the channel or chamber containing the gel to an appropriate temperature.
  • the magnetic particle mas ⁇ is transferred through the area of liquefication. The gel in at least this area should be at a vi ⁇ co ⁇ ity that permit ⁇ the particle ⁇ to move.
  • the magnetic particle ma ⁇ s through an air pocket 108 in the device 100 of Fig. 6, for example, in the channel 106 leading from the fir ⁇ t chamber 104 to the second chamber 78.
  • Medium containing ⁇ ample and magnetic particles i ⁇ introduced into device 100 through port 102.
  • the medium 114 containing the magnetic particle ma ⁇ s is subjected to a magnetic field gradient produced by magnet 118 to pull the magnetic particle ⁇ tas ⁇ out of the medium and into air pocket 108, which is usually an unwetted area 116 of the plastic film forming the chambers and/or channels of the device through which the particle ma ⁇ must move.
  • the magnetic particle ⁇ adhere to the inner pla ⁇ tic ⁇ urface of the channel and/or chamber.
  • a detergent such as a ⁇ , for example, Tween 20 ® (Triton X100 ® , lecithin) or the like.
  • a balance must be reached between elimination of adherence and increasing the wetting capability of the ⁇ urface by the detergent.
  • a relatively strong magnetic field gradient is necessary to move the magnetic particle mas ⁇ along the pla ⁇ tic surface in this embodiment.
  • Such a gradient should be about 5 to 15 KOe/cm and may require that the magnet be in contact with the pla ⁇ tic film a ⁇ the particle ⁇ move along. More than one magnet may be used to achieve a gradient sufficient to move the magnetic particle ma ⁇ , which should be as compact as pos ⁇ ible.
  • the magnet, and thus the magnetic particle mas ⁇ usually are moved at a speed of about 0.5 to 20 mm/sec.
  • the second chamber can contain reagents 88 for conducting amplification of the nucleic acid analyte if present.
  • reagents can be present in a liquid or, preferably, dry, form such as a tablet or powder and can include template dependent polynucleotide polymerase, deoxynucleotide tripho ⁇ phates, and polydeoxynucleotide primer( ⁇ ) .
  • the amplification can be conducted in thi ⁇ second chamber 78 or the content ⁇ of the ⁇ econd chamber can be transferred to the third chamber 84, where the amplification can be carried out.
  • Fluid communication is e ⁇ tabli ⁇ hed between ⁇ econd chamber 78 and a third chamber 84 u ⁇ ually by breaking a frangible seal 86, which can be broken by applying pressure to the contents of chamber 78 and forcing the contents into chamber 84.
  • the third chamber can contain reagent ⁇ 90 for detecting amplified nucleic acid or ⁇ uch reagent ⁇ can be introduced into the ⁇ econd or third chamber after amplification has been carried out.
  • a liquid containing the ⁇ e reagent ⁇ through a channel (not ⁇ hown) in fluid communication with chamber 84 wherein the fluid can be contained in a ⁇ eparate chamber in fluid communication with the channel and the channel may optionally be sealed with a frangible seal.
  • Fourth chamber 96 is in potential fluid communication with third chamber 90. At an appropriate time fluid communication is e ⁇ tabli ⁇ hed between chamber ⁇ 96 and 90 and the reaction mixture pa ⁇ e ⁇ into chamber 96 where, for example, ⁇ ignal i ⁇ read.
  • reaction conditions are chosen for carrying out the amplification reaction.
  • the following description sets forth such appropriate conditions, which are ⁇ ubject to modification by those skilled in the art depending on the specific reagents and other molecules chosen for any particular application.
  • an aqueous medium is employed.
  • Other polar co ⁇ olvent ⁇ may al ⁇ o be employed in the medium, u ⁇ ually oxygenated organic solvents of from 1-6, more usually from 1-4, carbon atoms, including alcohols, ethers and the like.
  • these cosolvent ⁇ are pre ⁇ ent in le ⁇ than about 70 weight percent, more u ⁇ ually, in less than about 30 weight percent.
  • the pH for the medium is usually in the range of about 5.5 to 10, more usually, in the range of about 6.5 to 9.5, and, preferably, in the range of about 7 to 9.
  • the pH and temperature are chosen and varied, as the case may be, so as to cause, either simultaneously or wholly or partially sequentially, di ⁇ ociation of any internally hybridized sequence ⁇ , hybridization of the primer with the single stranded polynucleotide sequences and extended primer once the primer has been extended, extension of the primer along the single stranded polynucleotide sequence ⁇ and extended primer, and di ⁇ ociation of the extended primer from its duplex.
  • Variou ⁇ buffere may be used to achieve the desired pH and maintain the pH during the determination.
  • Illustrative buffers include borate, pho ⁇ phate, carbonate, Tri ⁇ , barbital and the like.
  • Moderate temperature ⁇ are normally employed for carrying out the amplification.
  • the temperature employed are dependent on a number of con ⁇ ideration ⁇ ⁇ uch as, for example, the salt concentration and the pH of the medium, the solvent compo ⁇ ition of the medium u ⁇ ed, the length of the polynucleotide analyte and the length and nucleotide compo ⁇ ition of the primer(s) .
  • the medium is cycled between two or three temperatures.
  • the temperatures for the present method in conjunction with amplification generally range from about 10° to 105°C, more usually from about 40° to 99°C, preferably 50° to 98°C. Relatively low temperatures of from about 30° to 75°C can be employed for the hybridization steps, while denaturation and extension can be carried out at a temperature of from about 50° to 105°C.
  • the amplification i ⁇ conducted for a time sufficient to achieve a desired number of copies to achieve an accurate assay for a polynucleotide analyte is conducted for a time sufficient to achieve a desired number of copies to achieve an accurate assay for a polynucleotide analyte.
  • the time period for conducting the method is from about 20 seconds to 10 minutes per cycle and any number of cycles can be used from 1 to as high as 100 or more, usually 5 to 80, frequently 10-60. As a -matter of convenience it is usually desirable to minimize the time period and the number of cycles.
  • the time period for a given degree of amplification can be shortened, for example, by selecting concentrations of nucleoside triphosphate ⁇ ⁇ ufficient to saturate the polynucleotide polymerase and by increasing the concentrations of polynucleotide polymera ⁇ e and polynucleotide primer.
  • the time period for conducting the method i ⁇ from about 5 to 200 minutes.
  • Amplified nucleic acid can be detected in numerous ways.
  • molecules of the polynucleotide primer can be labeled with a reporter molecule such as a ligand, a small organic molecule including fluorescers, chemiluminescers and the like, catalysts, co-enzymes, radioactive sub ⁇ tance ⁇ , amplifiable polynucleotide ⁇ equences, a polypeptide, a support, an operator or the like.
  • a reporter molecule such as a ligand, a small organic molecule including fluorescers, chemiluminescers and the like, catalysts, co-enzymes, radioactive sub ⁇ tance ⁇ , amplifiable polynucleotide ⁇ equences, a polypeptide, a support, an operator or the like.
  • a reporter molecule such as a ligand, a small organic molecule including fluorescers, chemiluminescers and the like, catalysts, co-enzymes, radioactive sub ⁇ tance ⁇ , amplifi
  • the method comprise ⁇ treating the reaction mixture ⁇ u ⁇ pected of containing the amplified nucleic acid analyte under conditions ⁇ uch that the analyte, if pre ⁇ ent, causes a photosensitizer and a chemiluminescent compound to come into close proximity.
  • the photo ⁇ en ⁇ itizer generate ⁇ ⁇ inglet oxygen and activates the chemiluminescent compound when it is in clo ⁇ e proximity.
  • the activated chemiluminescent compound subsequently produces light.
  • the amount of light produced is related to the amount of analyte in the medium. More particularly, as applied to the present invention, the method comprises as a fir ⁇ t ⁇ tep providing a combination compri ⁇ ing the aforementioned medium suspected of containing amplified nucleic acid analyte bound to a particle which also has a chemiluminescent compound associated with the particle, a photo ⁇ en ⁇ itizer associated with a specific binding pair (sbp) member capable of binding to the amplified nucleic acid analyte.
  • the combination i ⁇ treated, usually by irradiation with light, to excite the photosensitizer, which is capable in it ⁇ excited state of activating oxygen to a singlet state.
  • the combination is then examined for the amount of luminescence or light emitted.
  • the amount of such luminescence is related to the amount of nucleic acid analyte in the medium.
  • the chemiluminescent compound is associated with an sbp member capable of binding amplified nucleic acid analyte and the particle to which the nucleic acid analyte is bound is associated with a photosensitizer.
  • Signal generated by the detection reagents i ⁇ generally measured or detected at the chamber where the reaction mixture is located. However, it is within the purview of the present invention to transfer the reaction mixture to a fourth chamber prior to reading the signal.
  • detection of signal can occur at the third chamber 84.
  • the chamber at which detection of signal occurs usually contains means for detection of the signal such a ⁇ , for example, an element capable of transmitting a signal, e.g., a window or electrode depending on the nature of the signal formed.
  • an optical signal is read through the wall of the chamber containing the reaction mixture.
  • kits can be used in accordance with the methods of the present invention in determining a polynucleotide analyte.
  • the kit comprises in packaged combination: (a) a device in accordance with the present invention and (b) particles capable of binding the nucleic acid analyte.
  • the kit can further include a labeled or unlabeled polynucleotide probe capable of binding to extended primer produced in the method of the invention.
  • kits above can further include in the packaged combination, if not included in the device, deoxynucleoside tripho ⁇ phate ⁇ (dNTP ⁇ ) such as, e.g., deoxyadenosine triphosphate (dATP) , deoxyguano ⁇ ine tripho ⁇ phate (dGTP) , deoxycytidine tripho ⁇ phate (dCTP) and deoxythymidine tripho ⁇ phate
  • dNTP ⁇ deoxynucleoside tripho ⁇ phate ⁇
  • dATP deoxyadenosine triphosphate
  • dGTP deoxyguano ⁇ ine tripho ⁇ phate
  • dCTP deoxycytidine tripho ⁇ phate
  • the kit can further include a polydeoxynucleotide polymerase and member ⁇ of a ⁇ ignal producing ⁇ ystem and also various buffered media, some of which may contain one or more of the above reagent ⁇ .
  • the relative amount ⁇ of the variou ⁇ reagent ⁇ in the kits can be varied widely to provide for concentrations of the reagents that substantially optimize the reactions that need to occur during the present method and to further substantially optimize the ⁇ ensitivity of any assay, in which the present method is employed.
  • one or more of the reagent ⁇ in the kit can be provided a ⁇ a dry powder, usually lyophilized, including excipients, which on dissolution will provide for a reagent solution having the appropriate concentrations for performing a method or assay in accordance with the present invention.
  • Each reagent can be packaged in separate containers or some reagents can be combined in one container where cro ⁇ -reactivity and ⁇ helf life permit.
  • the above discussion include ⁇ certain theories as to mechanisms involved in the present invention. These theories should not be construed to limit the present invention in any way, since it has been demonstrated that the present invention achieves the results described.
  • the above description and examples disclose the invention including certain preferred embodiments thereof. Modifications of the methods de ⁇ cribed that are obviou ⁇ to those of ordinary skill in the art such as molecular biology and related ⁇ cience ⁇ are intended to be within the scope of the following claims and included within the metes and bound ⁇ of the invention.

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Abstract

Dispositifs et procédés permettant d'effectuer un essai pour la détermination d'acides nucléiques. Les dispositifs comprennent (a) une chambre recevant l'échantillon, (b) un ensemble de chambres supplémentaires, dont au moins l'une contient des réactifs servant à effectuer un essai pour la détermination d'un acide nucléique, (c) un moyen de détection d'un signal produit par les réactifs, (d) un moyen permettant la communication par fluide entre les chambres supplémentaires, (e) un moyen permettant d'introduire un échantillon dans le dispositif, et (f) un moyen permettant de séparer l'analyte du reste de l'échantillon et de l'introduire dans l'une des chambres supplémentaires, ce moyen comprenant des particules pouvant être mises en suspension. Les procédés consistent à introduire un échantillon que l'on soupçonne contenir un analyte d'acide nucléique dans un dispositif du type décrit par la présente invention. L'analyte éventuellement présent et des particules dans un milieu sont transportés à travers les chambres du dispositif. Les réactifs servant à effectuer une amplification sont combinés avec le milieu transporté, qui est soumis à des conditions d'amplification de l'analyte. On examine ensuite le milieu afin de détecter la présence d'un analyte amplifié.
PCT/US1994/004849 1993-05-17 1994-05-03 Recipient de reaction pour essai par liaison specifique et procede d'utilisation WO1994026414A1 (fr)

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